PROJECT SUMMARY/ABSTRACT Plant terpenes are a critical source of clinically approved drugs and clinical candidates, yet very few complete biosynthetic pathways have been characterized. Due to the lack of efficient chemical synthesis routes, many complex plant natural product scaffolds including terpenes are currently still isolated from the producing plant or plant cell culture and then converted to a clinically-used drug by semisynthetic routes (e.g. digoxin and taxol on the 2015 WHO list of essential medicines). Lack of information regarding terpene biosynthetic pathways severely limits the use of promising new approaches to produce plant molecules in heterologous hosts (e.g. yeast strains that make artemisinin), as well as the intriguing possibility of engineering the biosynthetic pathways to access analogs and non-natural derivatives with greater efficacy. Given the critical role of medicinal plant terpenes in human health and utility of biosynthetic genes, we propose here systematic discovery of key medicinal plant terpene biosynthesis pathways, taxol and cyclopamine, and the engineering of yeast strains for scalable production. Classically, the discovery of plant pathways has been slower and more painstaking than bacterial pathways; however, our team has demonstrated two approaches that greatly accelerates identification of complete biosynthetic routes: (1) rapid combinatorial testing of enzymes in a N. benthamiana heterologous host, and (2) transcriptional profiling and co-expression analysis to identify pathway genes. This approach enabled the discovery of six enzymes that complete the pathway to the etoposide aglycone from the unsequenced medicinal plant Podophyllum in a matter of months and has also led to the discovery of numerous plant terpene pathways including a novel class of sesterterpenes. In this proposal, we have prioritized pathways for valuable medicinal plant terpenes that are notoriously difficult to access: the clinically used anticancer agent taxol and the clinical candidate cyclopamine. These compounds are representative medicinal plant terpenes that will be used to demonstrate the broad utility of our discovery and yeast engineering approach that can be applied for accessing many of the other >100,000 different plant terpenes in nature. In addition to yeast strains that produce these highly valuable plant terpenes, a major outcome of this work will be broadly applicable yeast synthetic biology tools for efficient production of multiple cytochromes P450s in series which represents a major bottleneck for efficient transfer of plant pathways to yeast heterologous hosts.